The Effects of Pesticides Mixture on Aquatic Organisms in Thailand

235 Pages·2015·2.11 MB·English

The Effects of mixtures of pesticides, in use
in Thailand, on the aquatic macrophyte
Lemna minor

Rungnapa Tagun
PhD

University of York
Environment
September 2014
Abstract

Abstract
The aim of the present study was to assess the effects of four herbicides commonly
used in Thailand (atrazine, 2,4-D, alachlor, paraquat)on the aquatic plant Lemna
minor under differing patterns of exposure (single-, mixture-, and sequential-
exposure). The endpoint of interest was the growth rate of plants over time.
In the single-compound toxicity studies, paraquat was found to be the most toxic
pesticide followed by alachlor, atrazine and 2,4-D. Mixture studies were then done
on the pesticides to understand how they would interact. Comparison of data from
toxicity tests on mixtures of the pesticides with modelling predictions indicated that
atrazine and2,4-D interact antagonistically whereas alachlor and paraquat interact
synergistically. These results are in agreement with other mixture studies with
pesticides.
Studies were also done to understand the effects of the different pesticides when
applied in sequence. Comparison of the experimental results with predictions from a
simple model demonstrated that at low effect concentration herbicides, the model
works well but at higher concentrations it falls down. To explore the reasons for this,
a further study was done to assess the carry-over toxicity of the study compounds.
This work demonstrates approaches to understand the effects of pesticides under
more realistic exposure conditions. It demonstrates that while modelling approaches
are available for estimating impacts under more realistic exposures, the accuracy of
the predictions is likely to be highly dependent on the mode of action and
concentration of the pesticide and the duration of the exposure.
List of contents

List of Contents
Abstract ........................................................................................................................ 2
List of Contents ............................................................................................................ 3
List of table .................................................................................................................. 7
List of figures ............................................................................................................. 10
Acknowledgments ...................................................................................................... 21
Author’s declaration ................................................................................................... 22
CHAPTER 1 .............................................................................................................. 23
1. Introduction ......................................................................................................... 23
Background and significance of the problems ....................................................................23
Agriculture and pesticide use in Thailand ...........................................................................24

Pesticidesin aquatic environment .......................................................................................28

Fate and behaviour of pesticides in aquatic environments. ...........................................28

The impacts of pesticides in aquatic environment .............................................................32

The impact of pesticides on aquatic organisms and ecotoxicological assessment of

pesticides on aquatic plant. ............................................................................................33
Aquatic macrophyte for risk assessment for pesticide .......................................................39

Pesticide mixtures in aquatic ecosystems and chemical interactions ................................40

Mixture toxicity theory ...................................................................................................40

Type of combined actions ...............................................................................................40

Experimental methods to assess pesticide mixture interactions ...................................42

The concept for calculating predictions with isobolographic methods ..........................42

Pesticide mixtures in aquatic ecosystems and previous studies into ecotoxicological

interactions of pesticide mixtures ......................................................................................46
Chronic and pulsed exposure of aquatic organisms to pesticides ..................................52

Modes of action / site of action of herbicides and type of damage on plants ...................54

Rationale for this study .......................................................................................................57

Aims and Objectives: ...........................................................................................................57

Test chemicals and test organism .......................................................................................58

Test chemicals .................................................................................................................58

The study pesticides ............................................................................................................61

3
List of contents

Atrazine ...........................................................................................................................61
2,4-D ................................................................................................................................61

Paraquat dichloride .........................................................................................................62

Alachlor ...........................................................................................................................62

Test organisms ................................................................................................................62

Environmental risk assessment of pesticides in Thailand ...................................................65

Europe (European Union) ...............................................................................................66

Structure of the Thesis ........................................................................................................68

Chapter 1 .........................................................................................................................68

Chapter 2 .........................................................................................................................68

Chapter 3 .........................................................................................................................68

Chapter 4 .........................................................................................................................69

Chapter 5 .........................................................................................................................69

Chapter 6 .........................................................................................................................69

CHAPTERII ............................................................................................................... 70

2. Survey of PesticidesUsed in Chiang Mai, Thailand............................................ 70
Introduction ........................................................................................................................70
The aim of this research ......................................................................................................72

Methodology .......................................................................................................................73

Study areas ......................................................................................................................73

Field sampling and data collection .................................................................................74

Assessment of aquatic exposure to pesticides in rice fields in Thailand ........................74

Results .................................................................................................................................75

Generalinformation .............................................................................................................75

Rice farming season ............................................................................................................76

Pesticides used ....................................................................................................................76

Exposure assessment for pesticides in rice fields in Thailand ............................................83

Discussion ............................................................................................................................83

Use of pesticide mixtures ................................................................................................85

Modelled herbicide concentrations in rice field .............................................................86

CHAPTER III ............................................................................................................ 87

3. The Effects of Mixtures of Herbicides on Lemna minor .................................... 87
Introduction ........................................................................................................................87

4
List of contents

Materials and Methods .......................................................................................................90
Chemicals ........................................................................................................................90

Test species and test conditions ..........................................................................................91

Lemna minor culture .......................................................................................................92

Single compound ecotoxicity tests. ................................................................................92

Results .................................................................................................................................98

Chemical analysis ............................................................................................................98

Discussion ..........................................................................................................................108

Single toxicity ................................................................................................................108

Mixture toxicity .............................................................................................................109

Conclusion .........................................................................................................................112

CHAPTER IV .......................................................................................................... 113

4. The Effects of Sequential Exposures to Multiple Herbicideson the Aquatic
MacrophyteLemna minor ......................................................................................... 113

Introduction ......................................................................................................................113
Materials and methods .....................................................................................................115

Chemicals ......................................................................................................................115

Lemna minor cultures ...................................................................................................115

Sequential exposure studies ..........................................................................................116

Short-term exposure .....................................................................................................116

Test conditions and observation of sequential toxicity ................................................118

Calculation of the measured and predicted growth rates ............................................119

Analytical methods .......................................................................................................121

Statistics ........................................................................................................................122

Results ...............................................................................................................................122

Toxicity of herbicides on Lemna minor based on the frond area .................................124

Dose response model for measured and predicted data .............................................141

Long-term sequential exposure ....................................................................................144

Discussion ..........................................................................................................................149

Conclusion .........................................................................................................................153

CHAPTER V ............................................................................................................ 155

5. The recovery potential pattern after short and prolonged exposure of Lemna
minor to herbicides ................................................................................................... 155

5
List of contents

Introduction ......................................................................................................................155
Material and method ........................................................................................................158

Plants and culturing ......................................................................................................158

Chemicals ......................................................................................................................159

Experimental method ...................................................................................................159

Statistical analysis .........................................................................................................162

Calculations of the average specific growth rate ..........................................................162

Chemical analysis ..........................................................................................................163

Results ...............................................................................................................................164

Chemical analyses .........................................................................................................164

Symptoms of herbicide toxicity (visible observe) .........................................................167

Short-term and long-term recovery patterns ...............................................................168

Discussion ..........................................................................................................................177

Conclusion .........................................................................................................................182

CHAPTER VI .......................................................................................................... 184

6. General Discussion............................................................................................ 184
Synthesis of the data from the three experimental chapters...........................................185
Risk of herbicide exposure in rice fields to the aquatic macrophyte Lemna minor .........188

Implications toward the risk of pesticides in Thailand’s environment .............................190

The limitations of this research ........................................................................................191

Conclusion .........................................................................................................................193

Appendix A .............................................................................................................. 195

Appendix B .............................................................................................................. 201
Appendix C .............................................................................................................. 207
Table C1: pH data of sequential exposure I (mean ±standard deviation for three
replicates) ..........................................................................................................................207
Table C1: (cont.) pH data of sequential exposure I (mean ±standard deviation for three

replicates) ..........................................................................................................................208
Appendix D .............................................................................................................. 210

Appendix E .............................................................................................................. 218
References ................................................................................................................ 223

6
List of tables

List of tables
Table 1-1:The most imported pesticide active ingredients in Thailand in the year
2000 (Sematong et al., 2008). Amounts are provided for the product and the
active ingredient. ................................................................................................ 25
Table 1-2: The most used pesticides in rural areas in Chiang Mai,Thailand (Panuwet
et al., 2008). ....................................................................................................... 27
Table 1-3: Pesticide persistence classification based upon degradation half-lives
(Kerle et al., 2007). ............................................................................................ 30
Table 1-4: The symptoms of phytoxicity in plants(European and Mediterranean
Plant Protection, 1997). ...................................................................................... 35
Table 1-5: List of ecotoxicity tests with aquatic organisms for the study compounds
investigated in this thesis. .................................................................................. 37
Table 1-6: Summary of pesticide mixture toxicity studies on aquatic organisms. .... 50
Table 1-7: Herbicide sites of action and injury symptoms to plant ........................... 55
Table 1-8: Physicochemicalproperties of atrazine, 2,4-D, paraquat and alachlor
(according Tomlin,2006).................................................................................... 59
Table 1-9: Data requirement and aquatic ecotoxicological risk assessment in
Japanese pesticide registration ........................................................................... 65
Table 1-10: Data requirement and aquatic ecotoxicological risk assessment in Europe
pesticide registration .......................................................................................... 66
Table 2-1: Pesticides used in Chiang Mai as recorded from a survey undertaken
during the period December 2011-January 2012. .............................................. 77
Table 2-2: Ranking of pesticide products in terms of annual quantity of active
ingredient used in the three districts studied in Chiang Mai. ............................. 79

7
List of tables

Table 2-3: Ranking of pesticides used based on active ingredient on paddy fields in
the Chiang Mai farms that were surveyed.......................................................... 80
Table 2-4: Frequency of pesticide application of small-scale farmers in Chiang Mai,
Thailand during December 2011. ....................................................................... 82
Table 2-5: Input values used for the first-tier PEC (predicted environmental
concentration) calculations in accordance with US-EPA (2007) ....................... 83
Table 3-1:Chemical characteristics and sites of action of the four herbicides used in
the present study (Tomlin, 2006) ....................................................................... 91
Table 4-1: Dosage of solvents and pesticide concentrations in different sequential
exposure studies. .............................................................................................. 118
Table 4-2: Experiment plan for short-term and long-term exposure to pesticides .. 119
Table 4-3: Analytical results for pulsed exposure studies and standard deviation. . 123
Table 4-4: The results of predicted models and actual observations in short-term and
long-term sequential exposure ......................................................................... 148
Table 5-1: The effective concentrations tested in the experiment of four herbicides
(µg/L) ............................................................................................................... 160
Table 5-2: Experiment plan and exposure durations of L. minor to four herbicides
.......................................................................................................................... 161
Table 5-4: Mean and standard deviations of growth rate at the end of test period .. 168
Table 5-5: Phytostatic and phytocidal concentrations of atrazine, 2,4-D, alachlor and
paraquat on L. minor in different exposure periods ......................................... 177
Table 6-1: Summary of the results from the studies of mixtures, short-term and long-
term sequential exposures, and recovery. ........................................................ 186
Table 6-2: input values used for risk quotient of aquatic macrophyte Lemna minor
.......................................................................................................................... 189

8
List of tables

Table F1: R2 and slope of short-term recovery based on ln(area)............................ 218

9
List of figures

List of figures
Figure 1-1: Summary of imported pesticides between 2000 to 2010 (Panuwet et al.,
2012a). ................................................................................................................ 25
Figure1-2:Isobologram showing antagonism, additive and synergism lines ............. 43
Figure 2-1:A map showing Mae Taeng, Mae Rim and San Patong districts which are
major rice producing areas in Chiang Mai province, Thailand. ......................... 73
Figure 2-2: General information of the farmers from Mae Taeng (MT), Mae Rim
(MR) and San Patong (SPT) districts, Chiang Mai province, Thailand during the
period December 2011-Januray 2012. ............................................................... 76
Figure 2-3: Amount of active ingredient (A.I.) in pesticide use at the three sites
studied from paddy fields in Chiang Mai. .......................................................... 81
Figure 3-1: pH value including mean and standard deviation (SD) (n=3) at day 0 and
day7 atrazine and 2,4-D mixture during the experiment.................................... 99
Figure 3-2:pH value including mean and standard deviation (SD) (n=3) at day 0 and
day7 alachlor and paraquat mixture during the experiment. ............................ 100
Figure 3-3: the percentage of recovery chemical analysis including mean and
standard deviation (SD) (n=3) of four herbicides. ........................................... 100
Figure 3-4: Dose response curve of atrazine and 2,4-D in single and mixture in each
ratio; atrazine in single test (3-4A), 2,4-D in single test (3-4B), atrazine:2,4-D
100:0 (3-4C) and atrazine:2,4-D 83:17 (3-4D) ................................................ 102
Figure 3-5: Dose response curve of atrazine and 2,4-D mixture each ratio;
atrazine:2,4-D 63:37 (3-5A) and atrazine:2,4-D 50:50 (3-4B), atrazine:2,4-D
37:63 (3-5C), atrazine:2,4-D 17:83 (3-5D), atrazine:2,4-D 0:100 (3-5E) ....... 103